Individuals with human immunodeficiency virus (HIV) experience brain pathology associated with greater motor/mood/cognitive disorders (collectively termed neuroAIDS) than the general population and these effects are exacerbated among opiate abusers. Moreover, gender differences exist with opiate-abusing women at greater risk to contract HIV compared to opiate-abusing men; yet, some women may experience lesser neuroAIDS symptomology than men once infection occurs. We recapitulated these effects in mice and have begun to elucidate the sex steroid hormones that interact with opiates to confer protection to neuroAIDS. The present proposal will begin to reveal the effects and mechanisms of sex steroid interactions with opiates and HIV in a translational approach that utilizes cultured murine and human neural cells, as well as transgenic whole-animal murine models. This Pathway to Independence Award (K99/R00) will begin to discern the cellular/molecular mechanisms by which sex steroid milieu may influence opiate/HIV interactions for neuroAIDS pathology. In the R00 independent phase of this grant, we will examine the protective effects of central steroid formation on opiate/HIV protein interactions in vivo using whole-animal models that conditionally-express central HIV-1 Tat (Aim 3a) or constitutively-express central gp120 (Aim 3b). Neurosteroid formation will be pharmacologically inhibited or facilitated in male and female mice and subjects will be assessed for neuroAIDS-like motor, affective, and cognitive behavior. Striatal tissue will be assessed for viable/degenerating neurons, astrocytes, and microglia via immunocytochemistry and viable striatal medium spiny neurons (MSNs) will be assessed for sublethal changes in morphology. The influence of pharmacodynamic targets that overlap between steroids, opiates, X4/R5-tropic HIV, and HIV proteins (Tat or gp120) will be examined in murine neural co-cultures obtained mice and differentiated fetal neural progenitor cells (Aim 3c). Cultures will be pretreated with pharmacological antagonists to NMDA receptors, mGluR1, estrogen receptors (alpha and beta), and mu opioid receptors. As in the prior K99 project (Aims 1-2), cultures will be assessed for MSN morphology, intracellular ion concentrations ([Ca2+]i and [Na+]i) within MSN soma and dendrites, and mitochondrial membrane potential (via JC-10 fluorescence imaging). Supernatants will be assessed for production of cytokines/chemokines, reactive oxygen species, and nitrites. This proposal enables the development of an innovative and independent research program.